U.S. patent application number 14/270414 was filed with the patent office on 2015-01-01 for methods for inhibiting prostate cancer by kinases and angiogenesis inhibitory mechanisms of ball moss extract.
The applicant listed for this patent is Joseph L. Bryant, Henry Lowe. Invention is credited to Joseph L. Bryant, Henry Lowe.
Application Number | 20150004260 14/270414 |
Document ID | / |
Family ID | 49670552 |
Filed Date | 2015-01-01 |
United States Patent
Application |
20150004260 |
Kind Code |
A1 |
Lowe; Henry ; et
al. |
January 1, 2015 |
METHODS FOR INHIBITING PROSTATE CANCER BY KINASES AND ANGIOGENESIS
INHIBITORY MECHANISMS OF BALL MOSS EXTRACT
Abstract
A method of treating prostate cancer by administering a
pharmaceutically-acceptable amount of a crude extract of the
indigenous Jamaican plant Ball Moss (Tillandsia Recurvata)
comprising one or more cycloartane isolates, and the isolates used
in the method for eliciting thereby a kinase inhibitory response of
prostate cancer cells by selectively inhibiting MRCK.alpha. kinase
and angiogenesis of (growth of new blood vessels) to reduce the
viability of prostate cancer cells. The method and compounds holds
promise as a chemopreventive agent, without causing excessive
damage to normal cells.
Inventors: |
Lowe; Henry; (West Indies,
JM) ; Bryant; Joseph L.; (Baltimore, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Lowe; Henry
Bryant; Joseph L. |
West Indies
Baltimore |
MD |
JM
US |
|
|
Family ID: |
49670552 |
Appl. No.: |
14/270414 |
Filed: |
May 6, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13910692 |
Jun 5, 2013 |
8715748 |
|
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14270414 |
|
|
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|
61655694 |
Jun 5, 2012 |
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Current U.S.
Class: |
424/725 ;
552/510 |
Current CPC
Class: |
A61K 36/88 20130101;
A61K 31/575 20130101 |
Class at
Publication: |
424/725 ;
552/510 |
International
Class: |
A61K 31/575 20060101
A61K031/575; A61K 36/88 20060101 A61K036/88 |
Claims
1. A compound for inhibiting prostate cancer, comprising a
pharmaceutically-acceptable amount of an extract of the indigenous
Jamaican plant Ball Moss (Tillandsia Recurvata) containing one or
more cycloartane isolates which elicit a kinase inhibitory and/or
angiogenesis inhibitory response of prostate cancer cells, without
causing excessive damage to normal cells.
2. A compound for treating a subject exhibiting a cancer cell
metastasis comprising a cycloartane phytochemical of the following
general chemical formula: ##STR00003## where R1 is R1 is OH, O2,
H.sub.2C or O.sub.2C, R2 is H, R3 is H.
3. The compound according to claim 2, wherein R4 is any one from
among the group consisting of the following formulae 2-9:
##STR00004##
4. The compound according to claim 2, wherein said cycloartane
phytochemical comprises a cycloartane type triterpenoid
compound.
5. The compound according to claim 4, wherein said cycloartane
phytochemical comprises any one or more from among a group
consisting of Cycloartane-3,24,25-triol, Cycloart-23-ene-3,25-diol,
Cycloart-25-ene-3,25-diol, 3,23-Dioxo-9,19-cyclolanost-24-en-26-ole
acid, Cycloeucalenol, 24,25-Dihydroxycycloartan-3-one,
Hydroxycycloart-23-en-3-one, 25, and sterculin A.
6. The compound of claim 2, wherein said cancer cell metastasis
comprises prostate cancer cells.
7. The compound of claim 6, wherein said compound reduces the
proliferation of prostate to cancer cells.
8. A method for inhibiting prostate cancer by the steps of:
administering a pharmaceutically-acceptable amount of an extract of
the indigenous Jamaican plant Ball Moss (Tillandsia Recurvata)
containing one or more cycloartane isolates to elicit a kinase
inhibitory and/or angiogenesis inhibitory response of prostate
cancer cells, without causing excessive damage to normal cells; and
eliciting with said extract a kinase inhibitory response of
prostate cancer cells without causing excessive damage to normal
cells.
Description
CROSS-REFERENCE TO RELATED APPLICATION(S)
[0001] The present application is a division of U.S. patent
application Ser. No. 13/910,692 filed on Jun. 5, 2013 which is
incorporated herein by reference. U.S. patent application Ser. No.
13/910,692 is in turn derives priority from U.S. Provisional Patent
Application Ser. No. 61/655,694, filed 5 Jun. 2012.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to anti-cancer drugs and, more
particularly, to a method for inhibiting prostate cancer by kinases
and angiogenesis inhibitory mechanisms of a class of cycloartanes
extracted from Ball Moss (Tillandsia Recurvata).
[0004] 2. Description of the Background
[0005] There is great growth in the overlapping fields of biology,
technology, and medicine, including remarkable advances in cellular
biology that have given a new understanding of the molecular basis
for some diseases. Nevertheless, the incidence of some forms of
cancer continues to rise. This is particularly true of prostate
cancer, a leading cause of death in men. Indeed, prostate cancer is
the second most frequently diagnosed cancer and sixth leading cause
of cancer death in males worldwide. A. Jemal et al., Global Cancer
Statistics, Ca Cancer J Clin 2011;61:69-90 (March-April 2011).
[0006] The three conventional treatment options include surgery,
radiation and chemotherapy, or some combination of the three.
Chemotherapy is the most widely used tool in prostate cancer
treatment especially where the cancer has metastasized or spread to
other organs of the body. However, conventional chemotherapy is not
specific to cancer cells and has serious toxic side effects.
Moreover, in recent years prostate cancer cells have exhibited
increased resistance to current chemotherapies. Consequently, there
exists an urgent need to discover and develop new therapeutics that
can slow the growth of cancer cells while having lesser side
effects on the patients. The search for new molecules to combat the
rising cases of prostate cancer especially those resistant to
current chemotherapy calls for urgent action.
[0007] The more recent development of target therapies for the
treatment of human cancers is revolutionizing the concept of cancer
treatment today and has led to a profusion of drugs to treat cancer
patients. These drugs include alkylating agents, intercalating
agents, antimetabolites, etc., most of which target DNA or enzymes
regulating the DNA duplication and elongation process. However,
rapidly growing tumors do not always exhibit high levels of cell
proliferation, but may also exhibit low levels of cell death
compared to the normal cell population from which these tumor cells
issue. For these types of rapidly growing tumors, the mentioned
drugs are not effective. In addition, like chemotherapy, the great
majority of the drugs currently available for treatment of cancer
are toxic and involve detrimental side-effects on healthy cells,
tissues and organs. Targeted anticancer therapies rely on compounds
that interfere with cellular targets, e.g., they inhibit molecular
targets that play a pivotal rote in tumor progression. Such
therapies specifically target the cancer cells, thus minimizing
toxicity. A number of such target-based anticancer therapies are
now successfully used in routine clinical practice.
[0008] A kinase is an enzyme that activates or inactivates certain
proteins that control several biological activities in the cell
including cell growth, proliferation, apoptosis (programmed cell
death) and metabolism. There are two main types of kinases: those
that phosphorylate tyrosine residues and those that phosphorylate
serine and/or threonine residues in target proteins. The latter,
protein kinase inhibitor's, may be categorized by the amino acids
whose phosphorylation is inhibited. The body uses kinases
extensively to transmit signals and control complex processes in
cells, and myriad different kinases have been identified in humans.
In the event of carcinogenesis, eukaryotic cells become predisposed
to rapid and uncontrollable growth. This is evidenced by elevated
levels of different kinases expressed in various cancers. It is for
this reason that kinase inhibitors hold promise in restoring normal
cell proliferation and may provide a key cancer treatment.
Consequently, in recent years kinase inhibitors have emerged as
major targets for therapeutics, particularly for cancer related
therapies. However, relatively few kinase inhibitors have been
identified to date and so effort is being made to discover new
molecules that interact with kinases. A number of kinase inhibitors
such as Gefitinib (Approved for Non-Small Cell Lung cancer) have
shown promise in clinical trials against prostate cancer. Most of
the other kinase inhibitors in clinical trial (3 in Phase III)
against prostate cancer targets kinases that inhibits angiogenesis.
Tumor angiogenesis is the formation of new blood vessels which
supplies cancer cells with nutrients and oxygen. Antiangiogenic
agents work by cutting off blood supply to tumors thus to starving
cancer cells to death.
[0009] A tyrosine kinase is an enzyme that can transfer a phosphate
group from ATP to a protein in a cell. Tyrosine kinase functions as
an "on" or "off" switch in many cellular functions. It can become
mutated, stuck in the "on" position, and cause unregulated growth
of the cell, which is a necessary step for the development of
prostate cancer. Numerous tyrosine kinase inhibitors have proven to
be effective anti-tumor agents and anti-leukemic agents. For
example, in chronic myelogenous leukemia, the Abelson tyrosine
kinase inhibitor Imatinib (Gleevec.TM.) targets the activity of
BCR-ABL oncoprotein; in acute promyelocytic leukemia (APL),
all-trans-retinoic acid (ATRA) or arsenic trioxide
(As.sub.2O.sub.3) targets PML-RAR.alpha. fusion. The introduction
of ATRA or Imatinib in the treatment of APL or chronic myelogenous
leukemia patients has significantly improved the management of
these diseases.
[0010] An effective tyrosine kinase inhibitor must be capable of
killing or incapacitating prostate cancer cells without causing
excessive damage to normal cells. Elevated levels of LIMK1 kinase
are expressed in prostate cancer, a kinase which itself is
activated by MRCK.alpha.. Inhibitors of the MRCK.alpha. kinase may
restore normal cell proliferation and provide a key solution to
cancer treatment. Medicinal plants have been one of the major
sources for the discovery of a number of current clinically used
anticancer drugs.
[0011] Jamaica is known for its rich biodiversity and its abundant
usage of medicinal plants as ethno medicines. Tillandsia recurvata
L. (Bromeliaceae) which is commonly called the Jamaican Ball Moss
or the Old Man's beard is one of the several important plants found
in Jamaica. Several countries report its use in their
ethnomedicine. The major reported use is in Brazil where the plant
is used against rheumatism, ulcers and hemorrhoids. Previous
phytochemical studies showed the presence of; five
hydroperoxyclycloartanes, a dicinnamate, a flavanone and a caffeic
acid ester from the whole plant extracts.
[0012] In his U.S. Pat. No. 7,713,556 issued May 11, 2010, one of
the inventors named herein investigated the anti-tumor and
anti-inflammatory properties of a silica acid moiety extract of
Jamaican Ball Moss.
[0013] The present inventors have found that a class of cycloartane
isolates from the indigenous plant selectively inhibits MRCK.alpha.
kinase and angiogenesis of (growth of new blood vessels) to reduce
the viability of prostate cancer cells. The extract holds promise
as a chemopreventive agent.
SUMMARY OF THE INVENTION
[0014] It is, therefore, the primary object of the present
invention to provide a method for inhibiting prostate cancer by
administering a pharmaceutically-acceptable amount of a crude
extract of the indigenous Jamaican plant Ball Moss (Tillandsia
Recurvata) containing one or more cycloartane isolates to elicit a
kinase inhibitory and/or angiogenesis inhibitory response of
prostate cancer cells, without causing excessive damage to normal
cells.
[0015] The selected class of cycloartane phytochemicals derived
from Jamaican Ball Moss produces promising anti-cancer and
anti-inflammatory activity by selectively inhibiting MRCK.alpha.
with greater potency than other potential chemotherapeutics, and
actually reduces the viability of several prostate cancer cell
lines.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Other objects, features, and advantages of the present
invention will become more apparent from the following detailed
description of the preferred embodiments and certain modifications
thereof when taken together with the accompanying drawings in
which:
[0017] FIG. 1 is the general structural formula of the cycloartane
phytochemicals according to the present invention.
[0018] FIG. 2 is the structural formula for the cycloartane
phytochemical Cycloartane-3,24,25-triol according to the
invention.
[0019] FIG. 3 is the structural formula for the cycloartane
phytochemical Cycloart-23-ene-3,25-diol to according to the
invention.
[0020] FIG. 4 is the structural formula for the cycloartane
phytochemical Cycloart-25-ene-3,25-diol according to the
invention.
[0021] FIG. 5 is the structural formula for the cycloartane
phytochemical 3,23-Dioxo-9,19-cyclolanost-24-en-26-oic acid
according to the invention.
[0022] FIG. 6 is the structural formula for the cycloartane
phytochemical Cycloeucalenol according to the invention.
[0023] FIG. 7 is the structural formula for the cycloartane
phytochemical 24,25-dihydroxycycloartan-3-one according to the
invention.
[0024] FIG. 8 is the structural formula for the cycloartane
phytochemical Hydroxycycloart-23-en-3-one,25 according to the
invention.
[0025] FIG. 9 is the structural formula for the cycloartane
phytochemical Sterculin A according to the invention.
[0026] FIG. 10 is a chart listing all twenty-two cycloartane type
triterpenoid compounds isolated from the crude extract.
[0027] FIG. 11 is a chart of the kd50 kinase interactions.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0028] The present invention is a therapeutic cycloartane extract
derived from plant biomass, and more specifically the indigenous
Jamaican plant Ball Moss (Tillandsia Recurvata), for use in
regressing cancerous tumors and/or for anti-inflammatory effect, as
well as the method for inhibiting prostate cancer by administering
a pharmaceutically-acceptable amount of the crude extract to elicit
a kinase inhibitory and/or anti-angiogenic response against
prostate cancer cells without causing excessive damage to normal
cells.
[0029] Preparation of the Ball Moss Extracts
[0030] The whole Tillandsia Recurvata plant was collected by hand
from trees and electricity poles at Kingston, Jamaica. A voucher
specimen of the plant was identified at the Institute of Jamaica
Herbarium where it is deposited with Accession Number: IJ 3411. The
collected plant material was air dried under shade, pulverized into
powder, and extracted, extracted at room temperature for 48 hours
in the dark with 95% ethanol followed by CH.sub.2Cl.sub.2.
Following the addition of water to the ethanol extract, a further
extraction was carried out in hexane. The hexane extract was
combined with the CH.sub.2Cl.sub.2 extract and evaporated to
dryness resulting in a green residue (8.55 g). 3.32 g of the
residue was fractionated by dry column flash chromatography on Si
gel using hexane/CH.sub.2Cl.sub.2/ethyl acetate and mixtures of
increasing polarity yielding 9 fractions (F1-F9). Degassing was
done by ultra-sonication of the samples to yield an insoluble
solid, which solid was then filtered. The fractions were subjected
to high performance liquid chromatography (HPLC) using a column
Phenomenex Luna.TM. C18, 5 .mu.m, 2.times.50 mm; eluent,
acetonitrile with 0.05% MeOH. This confirmed the presence of the
cycloartanes in all 9 fractions.
[0031] Select Class of Cycloartane Phytochemicals
[0032] The inventors undertook to identify the phytochemicals
responsible for the observed anti-cancer and anti-inflammatory
activities in ball moss and to further isolate these
phytochemicals. Identification of the various components was based
on liquid chromatography/mass spectroscopy (LC/MS) ions as
correlated to literature data. Several molecular weight matches
cycloartane compounds as well as a triterpene, sterculin-A, were
observed in the extracts as described below. Proton Nuclear
Magnetic Resonance Spectroscopy (1H-NMR) was performed and the
interpreted spectra were consistent with steroid-like
structures.
[0033] The identified compounds are in the general class of
cycloartane type triterpenoid compounds of Formula I (below), also
shown in FIG. 1:
##STR00001##
[0034] In Formula I, R1 is OH, O2, H.sub.2C or O.sub.2C, R2 is H,
R3 is H, and R4 is any one of Formulas 2-9:
##STR00002##
[0035] In all twenty-two cycloartane type triterpenoid compounds
were isolated in the crude extract. All twenty-two cycloartane type
triterpenoid compounds are illustrated in FIG. 10. Of these, eight
are shown in FIGS. 2-9, respectively, and are characterized as
follows:
[0036] FIG. 2: Cycloartane-3,24,25-triol
[0037] FIG. 3: Cycloart-23-ene-3,25-diol
[0038] FIG. 4: Cycloart-25-ene-3,25-diol
[0039] FIG. 5: 3,23-Dioxo-9,19-cyclolanost-24-en-26-oic acid
[0040] FIG. 6: Cycloeucalenol
[0041] FIG. 7: 24,25-Dihydroxycycloartan-3-one
[0042] FIG. 8: Hydroxycycloart-23-en-3-one,25,
[0043] FIG. 9: Sterculin A
[0044] Test Results
[0045] The crude Tillandsia Recurvata biomass extracted above were
tested in cell lines PC-3 and DU145 to determine their antiprostate
cancer activity as well as the interaction of the crude extract
against 451 kinases, as were the Cycloartane-3,24,25-triol and
Cycloart-23-ene-3,25-diol isolates. The cells (PC-3 and DU145) were
maintained in minimum essential media (MEM) supplemented with 10%
fetal calf serum (FCS), 20 mMl-glutamine, 2%
penicillin-streptomycin, and 0.2% gentamicin. Celts were maintained
at 37.degree. C. with 5% CO.sub.2 in Corning 75 cm3 culture flasks.
Cells were trypsinized and plated at the appropriate density
(500-2000 cells/well log phase 72 h post drug addition) into 96
well plates in media for approximately 18 h after which they were
exposed to cycloartanes; cycloart-23-ene-3,25-diol and cycloartane,
cycloart-23-ene-3,25-triol for 72 h. The compounds were solubilized
DMSO (>0.1%), Following the appropriate treatments, cell
proliferation was measured using the WST-1
(4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene
disulfonate) (Roche) colorimetric assay according to the
manufacturer's instructions. All assays were performed in
duplicates and were monitored spectrophotometrically at 450 nm/690
nm (Synergy.TM. HT 96-well Plate Reader--BIO-TEK). Cell viability
was assessed as percent of drugs relative to vehicle
solvent-treated control.
[0046] IC50 values were determined from the extract dose versus
control growth curves using Graph Prism software.
[0047] Competition binding assays were established, authenticated
and executed. For most assays, kinases were fused to T7 phage
strains and for the other assays, kinases were produced HEK-293
cells after which they were tagged with DNA for quantitative PCR
detection. In general, full-length constructs were used for small,
single domain kinases, and catalytic domain constructs for large
multi-domain kinases. The binding assays utilized
streptavidin-coated magnetic beads treated with biotinylated small
molecule ligands for 30 minutes at room temperature which generated
affinity resins for the kinase assays. The liganded beads were
blocked with excess biotin and washed with blocking buffer to
remove unbound ligand and to reduce non-specific phage binding.
Binding reactions were assembled by combining kinases, liganded
affinity beads, and test compounds in binding buffer. Test
compounds were prepared as 40.times. stocks in 100% DMSO and
diluted directly into the assay (Final DMSO concentration=2.5%).
All reactions were performed in polypropylene 384-well plates in a
final volume of 0.04 ml. The assay plates were incubated at room
temperature with shaking for 1 hour and the affinity beads were
washed. The beads were then re-suspended in elution buffer and
incubated at room temperature with shaking for 30 minutes. The
kinase concentration in the eluates was measured by quantitative
PCR and FIG. 11 presents kd50 for kinase interaction with the test
compound. These were determined using a standard dose response
curve using the hill equation. Curves were fitted using a
non-linear least square fit with the to Levenberg-Marquardt
algorithm.
[0048] The crude Ball Moss extract showed potent kinase inhibitory
activity against five kinases, four of which are implicated in
prostate cancer growth and spread. The Tillandsia Recurvata biomass
extract also shows antiangiogenic activity in an ex-vivo Rat Ring
Aorta assay. These results demonstrate that a
pharmaceutically-acceptable amount of 8-14 .mu.g/ml of the crude
extract of the indigenous Jamaican plant: Ball Moss (Tillandsia
Recurvata) elicits a pharmaceutically-effective kinase inhibitory
response of prostate cancer cells without causing excessive damage
to normal cells.
[0049] Similar results were observed with the two tested isolates,
and similar results are expected with the remaining isolates. These
results validate the anti-tumor activity of the ball moss crude
extract and its isolates, as well as their mechanism of action
being anti-angiogenesis which can also be deemed chemopreventive.
These results, makes crude Tillandsia Recurvata (Ball Moss) extract
containing any one or more of the above-identified isolates a very
good candidate for development into a prostate cancer treatment.
More studies are currently underway to confirm the ability of Ball
Moss to halt the growth of prostate tumors in mice as well as
measure the inhibitory effects of the crude extract on the 4
related kinases and antiangiogenic effects in vivo.
[0050] These natural isolates will prove useful in drug design
aimed at treating selected cancers with fewer side effects as well
as being anti-inflammatory agents. Of course, one skilled in the
art will understand that any of the cycloartane isolates described
above can be prepared by chemical synthesis as opposed to isolation
from their natural source, and the synthetic equivalents and their
analogs are also considered to be within the scope and spirit of
the invention.
[0051] Having now fully set forth the preferred embodiment and
certain modifications of the concept underlying the present
invention, various other embodiments as well as certain variations
and modifications of the embodiments herein shown and described
will obviously occur to those skilled in the art upon becoming
familiar with said underlying concept. It is to be understood,
therefore, that the invention may be practiced otherwise than as
specifically set forth in the appended claims.
* * * * *